Abstract

The purpose of this study was to characterize the role of M2 muscarinic receptors in inhibiting relaxant effects of drugs that stimulate cyclic AMP (cAMP) accumulation in the guinea pig ileum. We investigated the ability of oxotremorine-M (oxo-M) to inhibit cAMP accumulation in the presence of agonists that stimulate adenylyl cyclase in other cells and tissues. Appreciable stimulation of cAMP (>50% over basal levels) was achieved with forskolin and maximally effective concentrations of isoproterenol, cicaprost, prostaglandin E1, prostaglandin E2 and prostaglandin I2, with the stimulation over basal levels of cAMP being 14.9-, 2.51-, 2.45-, 2.27-, 2.28- and 1.52-fold, respectively. Moderate or no cAMP stimulation was observed with dopamine, 5-hydroxytryptamine, 5-methoxytryptamine, dimaprit, vasoactive intestinal peptide, SKF-38393, 2-chloroadenosine, CGS-21680, prostaglandin D2, secretin and vasopressin. Oxo-M (1 μM) inhibited cAMP accumulation by 35% under basal conditions. Oxo-M inhibited specific agonist-stimulated cAMP levels by 20 to 70%. However, oxo-M caused little or no inhibition of specific prostaglandin I2- and cicaprost-stimulated cAMP levels (5 and 0%, respectively). In general, there was a correlation between the abilities of the various agonists to stimulate cAMP accumulation and to cause relaxation of the isolated ileum, with an exception being cicaprost. Experiments were carried out with isolated ileum to determine whether activation of M2receptors inhibited the relaxant effects of the various agonists. In these experiments, the ileum was first treated withN-(2-chloroethyl)-4-piperidinyl diphenylacetate to selectively inactivate M3 receptors. After this treatment phase, contractile responses to oxotremorine-M were measured in the presence of histamine and a given relaxant agent. These measurements were repeated in the presence of the M2-selective antagonist AF-DX 116. Analysis of the data showed that part of the contractile response to oxotremorine-M could be attributed to an M2-mediated inhibition of the relaxation. This M2 component of the contractile response was greatest when forskolin or isoproterenol was used as the relaxant agent. In contrast, little or no M2 response was measured in the presence of dopamine and cicaprost.

This prediction has been borne out in studies on smooth muscle that had been treated with 4-DAMP mustard to inactivate most of the M3 receptors. Such treatment causes a 20- to 40-fold reduction in the potency of highly efficacious agonists when contractions are measured under standard conditions in the ileum and trachea (Thomas et al., 1993; Watson et al., 1995; Thomas and Ehlert, 1996). The large decrease in agonist potency can be attributed to the loss of M3 receptors. However, if contractions are measured in the same tissues in the presence of histamine and forskolin, a highly potent muscarinic response occurs that is blocked by selective antagonists in a manner consistent with an M2 response (Thomas et al., 1993; Thomas and Ehlert, 1996). Presumably, the mechanism for this contraction involves an M2-mediated inhibition of the relaxant effects of forskolin. This M2-mediated contractile response is pertussis toxin-sensitive, unlike the standard contractile response, which is insensitive to pertussis toxin (Thomas and Ehlert, 1994,1996). Using the same strategy, little or no contractile effects were detected for M2 receptors in the rat fundus and guinea pig esophagus (Thomas and Ehlert, 1996). M2 receptors have also been shown to inhibit the relaxant effects of isoproterenol on histamine-induced contractions of the ileum but not of the trachea (Thomas et al., 1993; Reddy et al., 1995; Watsonet al., 1995). The reason for this difference between relaxant agents is unclear but may be related to the greater ability of forskolin to increase cAMP levels, compared with isoproterenol. Other investigators have used a different technique to detect a role for the M2 receptor in contraction of the trachea (Fernandeset al., 1992; Watson and Eglen, 1994). However, see work byRoffel et al. (1993, 1995) for an opposing viewpoint.

It has long been known that isoproterenol is more effective at relaxing histamine-induced contractions, compared with those elicited by a muscarinic agonist (Van Amsterdam et al., 1989; Roffelet al., 1993). These observations have been interpreted as evidence that activation of M2 receptors inhibits the cAMP accumulation elicited by isoproterenol, thereby reducing its relaxant effects against M3-induced contractions, whereas histamine is without effect on cAMP levels. Alternatively, it has been proposed that cross-talk may occur between phospholipase C activation (via M3 receptor stimulation) and thebeta adrenergic signal transduction pathway (Roffel et al., 1993). A correlation between phosphoinositide hydrolysis and the shift in beta adrenergic receptor potency has been demonstrated (Van Amsterdam et al., 1989), and this effect may involve protein kinase C inactivation of Gs, resulting in functional uncoupling of beta adrenergic receptors (Pyneet al., 1992; Grandordy et al., 1994). Both M2 and M3 receptor-mediated antagonism of isoproterenol relaxant potency may be at work to varying degrees in different tissues, or a yet unknown response may be mediated by the M2 receptor. Regardless, the functional role of the M2 receptor in contraction has not been clearly defined in all smooth muscle tissues.

The purpose of the studies in the present report was to investigate the ability of the M2 receptor in the guinea pig ileum to inhibit both the cAMP accumulation and the relaxant effects of a variety of agents known to stimulate adenylyl cyclase in other cells and tissues. Our results are consistent with the postulate that the functional role of the M2 receptor in contraction depends on the amount of cAMP accumulation stimulated by the relaxant agent and the extent to which the M2 receptor inhibits the increase in cAMP.

Methods

cAMP accumulation.

Male Hartley guinea pigs (250–300 g) were sacrificed by asphyxiation with CO2, followed by exanguination. Their ilea were immediately dissected, and the longitudinal muscle was removed by the method of Rang (1964) and immediately placed in ice-cold KRB buffer (124 mM NaCl, 5 mM KCl, 1.3 mM MgCl2, 26 mM NaHCO3, 1.2 mM KH2PO4, 1.8 mM CaCl2, 10 mM glucose) gassed with O2/CO2 (19:1). Strips of muscle were cross-chopped at 350 μm with a McIlwain tissue chopper, washed extensively and equilibrated at 37°C for 15 min. In some experiments, tissues were incubated with the aziridinium ion of 4-DAMP mustard (40 nM) for 1 hr in the presence of AF-DX 116 (1 μM). After this incubation the tissue was washed extensively to remove 4-DAMP mustard and AF-DX 116. The slices were incubated in 10 ml of KRB buffer with [3H]adenine (1.0 μM, 50 μCi) for 40 min at 37°C, to allow uptake and incorporation into endogenous ATP. Slices were washed three times to remove extracellular [3H]adenine and were allowed to equilibrate for 15 min. Aliquots (70–100 μl) of gently packed tissue slices were incubated for 10 min at 37°C in KRB buffer (0.7 ml) containing 3-isobutyl-1-methylxanthine (0.5 mM) and various agonists. Ro2-01724 was substituted for 3-isobutyl-1-methylxanthine in experiments where 2-chloroadenosine or CGS-21680 was used, because of the ability of 3-isobutyl-1-methylxanthine to block adenosine receptors. Reactions were stopped by the addition of 0.3 ml of trichloroacetic acid (30%, w/v). Approximately 2000 cpm of [32P]cAMP was added to each sample as an internal standard. The tubes were centrifuged, and the [3H]cAMP and [3H]ATP were separated from the supernatant fraction using the chromatography method described by Salomon et al. (1974). The supernatant was applied to a cation exchange column (1.25 ml of Dowex AG 50W-X4, 200–400 mesh) and washed twice with 1.25 ml of water. This eluate was collected, and the radioactivity was measured as the incorporation of [3H]adenine into [3H]ATP. The Dowex column was positioned over a column of neutral alumina (0.6 g), and the [3H]cAMP was eluted onto the alumina column with 5 ml of water. The [3H]cAMP was eluted from the alumina with 4 ml of 0.1 M imidazole HCl (pH 7.5). This fraction was collected and the radioactivity was measured to determine the amount of accumulated [3H]cAMP. The [3H]cAMP values were corrected for recovery of the internal standard and are expressed as a percentage of the amount of incorporation of [3H]adenine, to correct for any variation in the amount of tissue added to each assay.

Isolated ileum.

Male Hartley guinea pigs were sacrificed as described above, and the whole ileum was rapidly removed. The most distal 10 cm of ileum was discarded and 2- to 3-cm ileal segments were cut, flushed with KRB buffer to remove ileal contents and mounted longitudinally in an organ bath containing KRB buffer at 37°C, gassed with O2/CO2 (19:1). Isometric contractions were measured with a force transducer and recorded on a polygraph and are expressed as the mass (grams) required to generate the measured force. The ileum was equilibrated for 1 hr at a resting tension equivalent to a load of 0.5 g. Three test doses of the muscarinic agonist oxo-M or histamine were added to the bath to ensure reproducibility of the preparation. Ileal segments that did not achieve >60% of the maximum from the test doses were discarded. Between each test dose the ileum was washed with fresh KRB buffer and incubated for 5 min. To calculate an EC50 value for oxo-M, 6 to 10 concentrations of the compound, spaced geometrically every 0.33 log units, were added cumulatively to the bath, and contractile responses were recorded. After an EC50 value for oxo-M was obtained, the ileum was washed and incubated for 30 min before additional measurements were made. In some experiments, tissues were incubated with the aziridinium ion of 4-DAMP mustard (40 nM) for 1 hr in the presence of AF-DX 116 (1 μM). Tissues were washed extensively to remove AF-DX 116 and unreacted 4-DAMP mustard. When an EC50 value for oxo-M was to be obtained in the presence of AF-DX 116, the antagonist was incubated with the ileum for 30 min before contractions were measured.

Formation of the aziridinium ion of 4-DAMP mustard.

4-DAMP mustard undergoes two sequential reactions in aqueous solution at neutral pH. The first of these is the cyclization to its reactive aziridinium ion, and the second is the hydrolysis of the aziridinium ion to the stable alcohol product. In all experiments in which it was used, 4-DAMP mustard (10 μM) was first incubated in 10 mM phosphate buffer (pH 7.4) at 37°C for 30 min, to allow formation of the reactive aziridinium ion (Thomas et al., 1992). Immediately after cyclization, the solution of the aziridinium ion was placed on ice and used immediately.

Data analysis.

Oxo-M inhibition of cAMP accumulation was calculated in two ways. Total inhibition (It) was calculated for each experiment with the following equation:It=OC−1×100Equation 1where O denotes the fold stimulation of cAMP in the presence of oxo-M and C denotes the fold stimulation of cAMP in the absence of oxo-M. The mean ± S.E.M. of the paired data are expressed in table 1. Inhibition of agonist-stimulated cAMP accumulation (Ia) was calculated with the following equation:Ia=O−boC−bc−1×100Equation 2where C and O are defined as above (mean values used), bo denotes the basal cAMP accumulation in the presence of oxo-M (mean of 0.65-fold) andbc denotes the basal cAMP accumulation in the absence of oxo-M (1.0-fold). The EC50 values of oxo-M (concentration of oxo-M required for half-maximal response) for contraction and inhibition of cAMP accumulation were estimated by nonlinear regression analysis of the data according to an increasing or decreasing logistic equation, as described previously (Candell et al., 1990). In some instances, a two-component model was used to analyze the contractile response data measured in 4-DAMP mustard-treated tissue in the presence of histamine and a relaxing agent. For this analysis, the following equation was fitted to the data by nonlinear regression analysis:y=MaxMaxH[oxo­M]H1[oxo­M]H1+EC50HH1+(1−MaxH)[oxo­M]H2[oxo­M]H2+EC50LH2Equation 3In this equation, Max denotes the maximal contractile response, MaxH denotes the proportion of the high-potency component, H1 and H2 denote the high- and low-potency Hill coefficients and EC50H and EC50L denote the EC50 values of the high- and low-potency components of the contractile response curve. The contractile response data measured in the absence of AF-DX 116 were analyzed according to equation 1. The data measured in the presence of AF-DX 116 were also analyzed according to equation 1, except with the high- and low-potency EC50 values multiplied by constants (shiftH and shiftL, respectively) corresponding to the factors by which AF-DX 116 increased the EC50 values. In this analysis, the data obtained in the presence and absence of AF-DX 116 were analyzed simultaneously, sharing the estimates of Max, MaxH,H1, H2, EC50H and EC50Lbetween the two sets of data. In most of the analyses, the estimates ofshiftH and shiftL were set at constants corresponding to the expected values for AF-DX 116 (1.0 μM) at M2 and M3 receptors (see “Results”).

Results

Muscarinic inhibition of agonist-stimulated cAMP accumulation.

We measured the ability of the highly efficacious muscarinic agonist oxo-M to inhibit the cAMP accumulation elicited by a variety of agents that have been shown to stimulate adenylyl cyclase in other tissues. The cAMP-stimulating agents were used at concentrations from 0.8 to 10 μM, whereas oxo-M was always used at a nearly maximally effective concentration of 1 μM. The largest increase in cAMP was elicited by forskolin (10 μM), which stimulated cAMP levels 14.9-fold, from a basal value of 0.35 ± 0.04% (expressed as a percentage of the total [3H]adenine-labeled nucleotides) to 5.35 ± 1.32%. Moderate stimulation of cAMP (2.51–1.52-fold stimulation over basal) was observed with isoproterenol (1 μM), PGE1 (10 μM), PGE2 (10 μM), cicaprost (0.8 μM) and PGI2 (10 μM), with stimulations over basal being 2.51-, 2.27-, 2.28-, 2.45- and 1.52-fold, respectively (table 1). Little or no cAMP stimulation was observed with dopamine, 5-HT, 5-MT, dimaprit, VIP, SKF-38393, 2-chloroadenosine, CGS-21680, prostaglandin D2, secretin and vasopressin. Oxo-M (1 μM) inhibited basal cAMP levels by 35%. Indomethacin and tetrodotoxin had no effect on the oxo-M inhibition of basal cAMP (data not shown). Oxo-M (1 μM) inhibited the cAMP measured in the presence of the various agents by 21 to 67% (total inhibition). PGI2 and cicaprost were exceptions, with oxo-M exerting total inhibition of only 12 and 17%, respectively. Because part of the total inhibition can be attributed to inhibition of basal cAMP levels, we also tabulated the percent inhibition of agonist-stimulated cAMP, as described in “Methods.” This percentage was calculated after the basal values in the absence (1.0-fold) and presence (0.65-fold) of oxo-M were subtracted from the agonist-stimulated values in the absence and presence of oxo-M, respectively (table 1). Such a calculation estimates the specific inhibition of each agonist effect by muscarinic receptor activation. Using this calculation, oxo-M specifically inhibited forskolin-, isoproterenol-, PGE1-, PGE2-, dopamine- and VIP-stimulated cAMP levels by 70, 38, 20, 29, 26 and 39%, respectively. PGI2-, cicaprost-, 5-MT- and dimaprit-stimulated cAMP levels were not specifically inhibited by oxo-M (0, 5, 0 and 0%, respectively).

The potency of oxo-M for inhibiting cAMP accumulation was estimated by measuring oxo-M concentration-response curves in the presence of either forskolin (10 μM), isoproterenol (1 μM), dopamine (10 μM) or cicaprost (0.8 μM) (fig. 1). To keep the experimental conditions the same as those of subsequent contractile studies, tissue was treated for 1 hr with 4-DAMP mustard plus 1 μM AF-DX 116 (see “Methods”) to inactivate M3 receptors selectively. Our group has previously shown that this treatment does not effect muscarinic inhibition of stimulated cAMP accumulation (Thomas et al., 1993). Oxo-M caused a concentration-dependent inhibition of the cAMP accumulation elicited by each of the stimulators. The potency of oxo-M for inhibiting the cAMP accumulation elicited by each of the agonists was approximately the same, yielding pEC50 values of 6.66, 6.80, 6.69 and 6.68 for forskolin, isoproterenol, dopamine and cicaprost, respectively (table 2). The maximal inhibition elicited by a high concentration of oxo-M varied with the agonist used to stimulate cAMP accumulation (table 2; fig. 1). The greatest maximal effect was observed with forskolin (72%), followed by dopamine (51%), isoproterenol (45%) and cicaprost (27%). In figure1, cAMP accumulation is plotted without subtraction of the basal values.

Estimates of the pEC50 and maximal effect of oxo-M inhibition of cAMP accumulation stimulated by different agents

Maximal relaxant effects of heterologous agonists.

We investigated the ability of each agonist to inhibit histamine-induced contractions in the guinea pig ileum, to identify those agents having a correlation between their stimulation of cAMP accumulation and their relaxant ability. The isolated ileum was contracted with histamine (0.3 μM) and allowed to stabilize, and then a maximal concentration of the cAMP-stimulating agent was added. The amount of relaxation recorded is expressed as a percentage of the original histamine contraction (table3). For the purpose of making the comparison, the agonists are listed in order of decreasing effectiveness in stimulating cAMP accumulation. In the rightmost column, the percentage relaxation value for each agonist is listed together with its rank order (in parentheses). The relaxant effects of forskolin, isoproterenol and cicaprost were rather large, representing 103, 97.5 and 43.9% inhibition, respectively, of the histamine-induced contraction. Dopamine, VIP, CGS-21680, 2-chloroadenosine and SKF-38393 caused a small amount of relaxation (13–30%).

Correlation of cAMP accumulation and relaxant efficacy of maximal concentrations of various receptor agonists

Figure 2 shows the relaxant effects of the various agents plotted against their stimulation of cAMP accumulation, expressed relative to basal levels (basal cAMP = 1.0). It can be seen that there is a general correlation between the ability of agents to stimulate cAMP accumulation and to relax histamine-induced contractions. These data indicate that the ability of an agonist to stimulate cAMP accumulation is predictive of its relaxant efficacy (fig. 2). One notable exception is cicaprost (0.8 μM), which caused a large increase in cAMP accumulation (2.45 ± 0.25-fold) but only a modest amount of relaxation (43.9 ± 5.43%). It may be that this prostanoid derivative has a small contractile component to its action that partially opposes a large cAMP-mediated relaxation, resulting in a net relaxation much less than that expected from its cAMP response. If the data for cicaprost are excluded, figure 2 suggests that nearly complete inhibition (97%) of 0.3 μM histamine-induced contractions occurs when cAMP levels are increased approximately 2.5-fold over basal levels (e.g., isoproterenol at 1 μM) and that the increase in cAMP accumulation elicited by forskolin (14.9-fold) greatly exceeds that required to cause complete relaxation.

Comparison of cAMP accumulation (fold stimulation over basal level) and relaxant activity (percent relaxation of histamine-induced contraction) of maximal concentrations of various receptor agonists. Each data point represents a mean of 3 to 10 experiments.

Relaxant effects of agonists on muscarinic and histamine-induced contractions.

The potencies of the various agents for inhibiting histamine- and oxo-M-mediated contractions were compared. Only those agents that demonstrated significant relaxant effects (forskolin, isoproterenol, dopamine and cicaprost) were investigated. For these experiments, the isolated ileum was precontracted with either histamine (0.3 μM) or oxo-M (40 nM). These concentrations were chosen because they reliably produced contractions of equal size. Over the course of these studies, the estimates of the average tension ± S.E.M. (expressed in units of mass) elicited by histamine and oxo-M were 3.20 ± 0.22 g and 3.33 ± 0.20 g, respectively (not statistically different, P = .67). Cumulative concentration-relaxation curves were measured for each relaxing agent against histamine and oxo-M (fig. 3). The EC50 value and maximal relaxant effect for each agent are listed in table 4. In general, each agent was more effective at inhibiting histamine-induced contractions, compared with oxo-M-induced contractions. At high concentrations, forskolin caused complete inhibition of both histamine- and oxo-M-induced contractions. In contrast, the maximal relaxant effect of the other agents was always much less when oxo-M was used as the contractile agent, compared with histamine.

Concentration-response curves for relaxation by various agents (A–D) against contractions induced by 0.3 μM histamine (○) and 40 nM oxo-M (•). Data are presented as the percent decrease in tension of the initial contraction. Each point represents the mean ± S.E.M. of three experiments.

Relaxant potencies of various agents against histamine- and oxo-M-induced contractions

Contractile responses in 4-DAMP mustard-treated ilea.

To determine whether the M2 receptor could elicit an indirect contraction by preventing the relaxant effects of the various cAMP-stimulating agents, we measured contractile responses to oxo-M in isolated ileum according to the experimental design shown in figure4. In the first phase of the experiments (treatment phase), the ileum is incubated with 4-DAMP mustard (40 nM) in the presence of AF-DX 116 (1 μM) for 1 hr and then washed extensively (Thomas et al., 1993). This treatment inactivates most of the M3 receptors without affecting the M2receptors. In the second phase (test phase), the ileum is contracted with a nearly maximally effective concentration of histamine, and then a relaxant agent is added. After a stable relaxation is achieved, increasing concentrations of oxo-M are added to the bath so that cumulative concentration-response curves can be measured. These test phase measurements are repeated in the presence of AF-DX 116 to characterize the muscarinic receptor subtypes mediating the contraction. Experiments carried out under these conditions are shown in figure 5, B to E. In figure 5, the horizontal lines denote the size of the contraction elicited by histamine (0.3 μM) alone (upper line) and in combination with the relaxant agent (lower line). A summary of EC50 values and Hill coefficients is found in table 5.

Effects of 1 μM AF-DX 116 on estimates of the EC50 and Hill coefficient for oxo-M-induced contractions under standard conditions and conditions of elevated cAMP

For purposes of comparison, we first measured contractions under standard conditions, i.e., without first contracting with histamine and relaxing with a relaxant agent. Under these conditions, oxo-M contracted the ileum with an EC50 value of approximately 22 nM. This contractile response is mediated by the M3 receptor, as demonstrated by several investigators (Candell et al., 1990; Eglen and Harris, 1993). After 4-DAMP mustard treatment, oxo-M was still capable of eliciting a response under these conditions, but with 9-fold lower potency. Figure 5A shows the contractile responses to oxo-M under standard conditions after 4-DAMP mustard treatment. Under these conditions, AF-DX 116 (1.0 μM) caused a 2.8-fold increase in the EC50 value of oxo-M. This degree of shift is consistent with an M3 receptor-mediated contraction, as demonstrated by application of the simple competitive inhibition relationship:KB=[AF­DX116]CR−1Equation 4in which KB denotes the dissociation constant of AF-DX 116, and CR denotes the shift in the concentration-response curve (i.e., EC50 value of oxo-M in the presence of AF-DX 116 divided by that measured in the absence of AF-DX 116). Equation 4 yields an estimate of 6.26 for the pKB value of AF-DX 116, which is in excellent agreement with the binding affinity (Kd value) measured in Chinese hamster ovary cells transfected with the M3 subtype of the muscarinic receptor (6.10) (Esqueda et al., 1996). When forskolin (10 μM) was used as the relaxing agent, AF-DX 116 caused a 25-fold rightward shift of the oxo-M concentration-response curve, increasing the EC50 from 16.3 nM to 473 nM and causing a change in the Hill coefficient from 1.0 to 1.9 (table 5; fig. 5B). This degree of shift yields an estimate of 7.38 for the pKB value of AF-DX 116, which is in close agreement with the Kd value of AF-DX 116 measured in Chinese hamster ovary cells transfected with the M2 subtype (7.27) (Esqueda et al., 1996). These results indicate that the M2 receptor mediates the contractile response in the presence of histamine and forskolin after 4-DAMP mustard treatment. When tested in the same experimental paradigm but with other relaxant agents, AF-DX 116 usually caused shifts intermediate between those expected for M2 receptor- and M3 receptor-mediated contractions (table 5; fig. 5, C–E), suggesting that both M2 and M3 receptors contribute to the contraction. However, when cicaprost was used as the relaxant agent, the antagonistic effect of AF-DX 116 (2.7-fold shift) was consistent with that expected for an M3 response, with little or no contribution of the M2 receptor.

The biphasic nature of the control isoproterenol curve (fig. 5C) is consistent with this notion. Therefore, we analyzed this curve according to a two-component model, by nonlinear regression analysis, as described in “Methods.” Regression analysis showed that the data were consistent with a two-component model having high- and low-potency EC50 values of 15.5 and 736 nM, respectively. The high-potency component accounted for 54% of the overall maximal response. In the presence of AF-DX 116 (1.0 μM), the high- and low-potency EC50 values were shifted 17.2- and 1.1-fold, respectively. These shifts are very similar to those expected for antagonism of M2 (20-fold) and M3 (2.26-fold) responses, assuming pKd values of 7.27 and 6.1 for AF-DX 116 at M2 and M3receptors, respectively (see eq. 4). Because errors in the estimates of parameters by nonlinear regression analysis are correlated, we analyzed each curve by setting the shift values for the high- and low-potency components equal to values expected for antagonism of M2-mediated (20-fold) and M3-mediated (2.3-fold) responses, respectively. A summary of this analysis appears in table 5. The contribution of the M2 component was greatest with forskolin (100%, 1.8 g) as the relaxant agent, intermediate with isoproterenol (39%, 1.6 g) or dopamine (38%, 0.7 g) and least with cicaprost. The data with cicaprost were not analyzed by a two-site model, because the antagonistic effect of AF-DX 116 (2.7-fold shift) was approximately the same as that observed under standard conditions (2.8-fold), consistent with the response being entirely mediated by the M3 receptor.

Discussion

Reddy et al. (1995) have demonstrated that a variety of agents, including beta adrenergic agonists and prostaglandins, stimulate cAMP accumulation in slices of the longitudinal muscle of the guinea pig ileum. Those investigators measured the cAMP response to each agonist at a concentration of 10 μM and in the presence of a low concentration of forskolin (0.1 μM), which enhanced the cAMP response. Among the compounds tested, PGE1 elicited the largest increase in cAMP over basal levels (5.3-fold). Intermediate increases in cAMP were noted with PGE2 (3.4-fold) and isoproterenol (3.0-fold), whereas the smallest increases were observed with VIP (2.3-fold), 5-HT (1.7-fold) and the beta-3-selective agonist BRL 37344 (1.4-fold). In the present study, we observed a similar pattern of cAMP stimulation with these agonists, although the absolute stimulation was about 50% less, on average. This decrease can be attributed to the lack of forskolin (0.1 μM) in our assays, because Reddy et al.reported that forskolin enhanced the cAMP response to isoproterenol (10 μM) by 40%. We also observed appreciable stimulation of cAMP by the prostanoid derivative cicaprost (2.45-fold), PGI2(1.52-fold), the H2 histamine agonist dimaprit (1.32-fold) and 5-MT (1.25-fold).

When measured in the presence of the highly efficacious muscarinic agonist oxo-M (1.0 μM), the cAMP accumulation elicited by the various agonists was substantially inhibited. Presumably, this muscarinic inhibition is mediated by the M2 receptor because, in both rat and guinea pig ileum, subtype-selective antagonists block the inhibition in a manner consistent with an M2 response (Candell et al., 1990; Griffin and Ehlert, 1992; Reddyet al., 1995). In the present study, both basal and agonist-stimulated cAMP accumulation was inhibited by oxo-M. To estimate that component of the muscarinic inhibition that could be attributed to specific inhibition of agonist-stimulated cAMP accumulation, we subtracted the appropriate basal values from the data, as described in “Methods,” before estimating the percentage inhibition. These calculations yielded an estimate of 70% for the inhibition of forskolin-stimulated cAMP accumulation by oxo-M. In contrast, oxo-M inhibited the cAMP accumulation elicited by isoproterenol, PGE1, PGE2, dopamine, 5-HT and VIP to a lesser extent (38, 20, 29, 26, 23 and 39%, respectively) and was without effect on the cAMP accumulation elicited by cicaprost, PGI2, 5-MT or dimaprit. Thus, activation of M2muscarinic receptors inhibits the increase in cAMP elicited by almost all of the agonists tested. In the rat ileum, M2 receptors mediate inhibition of forskolin- and isoproterenol-stimulated cAMP accumulation but not that elicited by PGE1 or PGE2 (Griffin and Ehlert, 1992). The different responses of these two tissues may be due to the greater abundance of M2receptors in the guinea pig ileum (Michel and Whiting, 1990; Candellet al., 1990).

In addition to cAMP, other mediators elicit relaxation in smooth muscle, including cyclic GMP, which may be stimulated either directly or indirectly (through nitric oxide synthase pathways). To identify agents that cause relaxation through cAMP, we measured the ability of the agents listed in table 1 to inhibit histamine-induced contractions, so that it would be possible to identify agents showing a positive correlation between cAMP accumulation and relaxation. Among the compounds tested, only forskolin, isoproterenol, cicaprost and dopamine caused appreciable relaxation. CGS-21680, 2-chloroadenosine, SKF-38393 and VIP elicited small inconsistent relaxations and other agents, such as PGE1, PGE2, PGI2, 5-HT, 5-MT and dimaprit, caused small contractions, presumably due to their respective abilities to activate multiple receptor subtypes, including some coupled to phosphoinositide hydrolysis. Among the relaxant agonists, there was a general correlation between the potential of agonists to stimulate cAMP and their ability to cause relaxation, suggesting that cAMP is the predominant mechanism for relaxation (fig. 2). One notable exception was cicaprost, the prostanoid IP receptor-selective agonist, which caused proportionately greater cAMP accumulation than relaxation.Lawrence et al. (1992) characterized prostanoid receptors in the guinea pig ileum and found that the IP receptor is implicated in direct smooth muscle relaxation and, to a lesser extent, neuronal stimulation of acetylcholine release. Indeed, in our experiments with cicaprost, a small contractile component frequently preceded relaxation. This effect accounts for the fact that cicaprost caused less relaxation than predicted from its potential to stimulate cAMP accumulation.

It is well established that isoproterenol is more effective at inhibiting histamine-induced contractions in the trachea, compared with those elicited by muscarinic agonists (Van Amsterdam et al., 1989; Roffel et al., 1993). To explain the differential effects of isoproterenol, Torphy et al. (1983) suggested that muscarinic inhibition of adenylyl cyclase protects the muscarinic contractile mechanism from the cAMP-mediated relaxant effects of isoproterenol. It is now known, of course, that inhibition of adenylyl cyclase and stimulation of contractions are mediated by two different muscarinic receptors (i.e., M2 and M3 receptors, respectively) (Peralta et al., 1988; Candell et al., 1990). Histamine lacks this two-pronged mechanism, so its contractile response is more sensitive to the relaxant effects of isoproterenol. Although direct evidence for a role of the M2 receptor in inhibiting the relaxant effects of isoproterenol on M3-mediated contractions in the trachea and ileum has been obtained by several investigators (Thomas et al., 1993; Watson et al., 1995), it is unclear whether the M2 mechanism is the only mechanism that accounts for the differential relaxant effects of isoproterenol (Roffel et al., 1995). We chose to compare the relaxant potential of the various cAMP-stimulating agents against histamine- and oxo-M-induced contractions, to obtain a simple measure of whether the M2receptor diminished the relaxant effect of the agent. If an agent was more effective at inhibiting histamine-induced contractions, compared with those elicited by oxo-M, then a role for the M2receptor might be inferred. The greatest differential relaxant effects were noted with forskolin, isoproterenol and dopamine, whereas little or no discrimination was noted with cicaprost (fig. 3). Oxo-M inhibition of cAMP levels stimulated by each of these agents (table 1) followed a similar pattern. Thus, there appears to be at least a rough correlation between the extent to which the M2 receptor inhibits the cAMP accumulation elicited by a drug and the extent to which the drug displays differential relaxant effects.

In examining the data shown in figure 3, it becomes apparent that there is no simple way to quantify the differential relaxant effects of the various agents. Forskolin exhibited a 6-fold difference in relaxant potency between histamine and oxo-M but no change in maximal response, whereas the other agents showed differences in both potency and maximal response. If the mechanism for the reduced relaxant activity in the presence of oxo-M is caused by M2-mediated inhibition of cAMP accumulation, then, in the presence of oxo-M, relaxing agents should behave as if they are less efficacious at stimulating cAMP accumulation, and hence relaxation. A useful measure of the apparent decrease in intrinsic efficacy is the degree of receptor inactivation that would cause an equivalent loss of activity. We have used the term “stimulus inactivation” to denote this decrement in relaxant activity against oxo-M-induced contractions, and we have described a means of calculating this parameter in the “.” Table 4lists our estimates of this parameter. It can seen that there is a rough correlation between the ability of oxo-M to inhibit both the relaxant stimulus and the cAMP accumulation elicited by the various agents. Interestingly, this correlation becomes closer if the inhibition of cAMP accumulation is calculated by subtracting the control basal value (1.0-fold) from both the stimulated and inhibited values before calculating the percentage inhibition by oxo-M. When calculated in this manner, the oxo-M inhibition of the cAMP accumulation stimulated by forskolin (73%), isoproterenol (61%), dopamine (110%) and cicaprost (29%) is in agreement with the degree to which oxo-M inhibits the relaxant stimulus generated by the agents (82, 68, 93 and 4%, respectively).

As described above, forskolin was able to cause complete inhibition (100%) of both histamine- and oxo-M-induced contractions, whereas isoproterenol, dopamine and cicaprost showed reduced maximal responses against oxo-M, relative to histamine. This behavior can be rationalized with the relationship between relaxation and cAMP levels shown in figure 2. It can be seen that complete relaxation (100%) of histamine-induced contractions occurs when cAMP levels are stimulated approximately 2.5-fold over basal. Even though oxo-M inhibited forskolin-stimulated cAMP levels by 70%, the residual level of cAMP in the presence of forskolin and oxo-M (4.5-fold over basal) (table 1) still exceeded that required for maximal relaxation. In contrast, the cAMP levels elicited by isoproterenol, dopamine and cicaprost in the presence of oxo-M (1.59-, 0.96- and 2.03-fold, respectively) (table 1) were insufficient to cause complete relaxation.

To further explore the role of the M2 receptor in opposing smooth muscle relaxation, we used the novel method outlined in figure4, which was previously developed in our laboratory (Thomas et al., 1993). We first inactivated M3 receptors with the irreversible alkylating agent 4-DAMP mustard and then measured responses to a muscarinic agonist in the presence of histamine and a relaxing agent. When forskolin was used as the relaxant agent, theKB values of AF-DX 116, methoctramine,p-fluorohexahydrosiladifenidol and pirenzepine were in good agreement with their respective binding affinities for native and cloned M2 receptors but much different from those reported for the M1, M3, M4 and M5 subtypes (Ehlert and Thomas, 1995; Esqueda et al., 1996). Thus, under the conditions of the experiment, oxo-M acts through the M2 receptor to release the brake that forskolin has put on histamine-induced contractions. Thomas et al. (1993) and Reddy et al. (1995) have used this strategy to show that the M2 receptor also opposes the relaxant effect of isoproterenol on histamine-induced contractions in the guinea pig ileum. In this study, we have reexamined forskolin and isoproterenol, so that data on contractions and cAMP accumulation could be compared for a group of relaxant agents.

When forskolin was used as the relaxing agent under the experimental conditions shown in figure 4, AF-DX 116 (1.0 μM) shifted the concentration-response curve for oxo-M to the right 25-fold, in a manner consistent with simple competition at an M2muscarinic receptor. The calculated KB value (7.38) was nearly the same as the estimate of the binding affinity of AF-DX 116 for the cloned M2 receptor (7.27) when measured in modified KRB buffer (Esqueda et al., 1996). The maximal response of oxo-M was only about 55% of that of the histamine-induced contraction, suggesting that the M2 receptor cannot fully oppose the inhibitory effect of forskolin on histamine-induced contractions. When isoproterenol was used as the relaxant agent during the test phase, the concentration-response curve of oxo-M had a low slope and appeared biphasic, suggesting high- and low-potency components. AF-DX 116 simplified the curve by causing a greater apparent competition at the high-potency component. We reasoned that at low concentrations oxo-M was acting through the AF-DX 116-sensitive M2 receptor to disinhibit histamine-induced contractions, whereas at high concentrations oxo-M might be eliciting contractions through M3 receptors not inactivated by 4-DAMP mustard. The contribution of the M3 receptor could account for the large maximal response, which frequently exceeded the histamine-induced contraction, although these did not differ statistically. Consequently, we analyzed the data according to a two-component model, assuming that AF-DX 116 (1.0 μM) should cause shifts of 20- and 2.3-fold in the M2 and M3 components of the concentration-response curve, respectively. Regression analysis yielded estimates of the size of the M2 component when the different relaxing agents were used. The size of the M2component of the contractile response appears to be related to the amount of the relaxation induced by the specific relaxant agent and the extent to which the M2 receptor inhibited the increase in cAMP elicited by the agent. When expressed in mass equivalents, the maximal contractile responses of the M2 components were 1.8 and 1.6 g when forskolin and isoproterenol were used as relaxing agents, respectively. No M2 component was apparent when cicaprost was used as the relaxant agent, in agreement with the one-site analysis, where the shift induced by AF-DX 116 was in close agreement with an M3 response. These estimates correlate roughly with the extent to which oxo-M inhibited the cAMP accumulation elicited by forskolin (70%), isoproterenol (38%) and cicaprost (5%). Although oxo-M caused a 26% inhibition of dopamine-stimulated cAMP accumulation, dopamine elicited a relatively small relaxation, so that the M2 component of the response was only 0.7 g. Collectively, these data show general agreement between the extent to which activation of the M2 receptor opposes the ability of a drug to stimulate cAMP accumulation and its ability to inhibit histamine-induced contractions.

When forskolin was used as the relaxing agent during the test phase, the concentration-response curve of oxo-M was relatively simple and lacked the low-potency M3 component characteristic of the data collected with the other relaxant agents. It seems likely that the higher levels of cAMP elicited by forskolin may have completely suppressed M3-mediated contractions already greatly inhibited by 4-DAMP mustard.

The potency of oxo-M for eliciting contractions through the M2 mechanism is very high. More specifically, when measured during the test phase in the presence of different relaxant agents, the EC50 values of the M2 component were in the range of 8 to 16 nM. In contrast, the EC50 value of oxo-M for eliciting contractions through the M3 receptor under standard conditions was approximately 30 to 40 nM. Moreover, after 4-DAMP mustard treatment, the EC50 value of the standard contractile response was increased to 207 nM. Thus, even after the M3 response has been greatly suppressed, oxo-M can elicit contractions through the M2 mechanism with about 20-fold greater potency, underscoring the significance of this phenomenon.

When measured under the conditions of the test phase, M2-mediated contractions were most easily detected when forskolin was used as the relaxing agent. Under these conditions, the contractile response to oxo-M yielded a relatively simple concentration-response curve that was shifted to the right 25-fold in the presence of the M2-selective antagonist AF-DX 116 (1.0 μM). When the other relaxant agents were used, the M3receptor contributed to the contractile response, thereby diminishing the relative contribution of the M2 receptor and making it more difficult to detect the M2 component of the response. These observations correlate generally with our measurements of cAMP. Forskolin caused by far the largest increase in cAMP accumulation, and oxo-M inhibited forskolin-stimulated cAMP levels by 70%. In contrast, oxo-M inhibited isoproterenol-stimulated cAMP accumulation by only 38% and the cAMP levels elicited by the other agents by less. We previously suggested (Thomas and Ehlert, 1996) that these observations might explain, in part, why it was possible to measure an M2-mediated inhibition of the relaxant effect of forskolin, but not isoproterenol, on histamine-induced contractions of the trachea (Watson et al., 1995). Detecting a role for the M2 receptor in inhibiting the relaxant effects of isoproterenol in the trachea may be easier when more M3receptors are selectively inactivated. Regardless, M2receptors have been shown to inhibit the relaxant effects of forskolin on M3-mediated contractions of the trachea (Thomas and Ehlert, 1996). Differences in the contractile effects of the M2 receptor in different tissues might also be explained by a variation in the density and coupling efficiency of the receptor. A decrease in the latter might account for our inability to measure an M2-mediated inhibition of the relaxant effect of forskolin on histamine-induced contractions of the rat fundus and guinea pig esophagus (Thomas and Ehlert, 1996).

In summary, our results confirm that the M2 receptor has the role of inhibiting the relaxant effects of agents that increase cAMP in the guinea pig ileum. The involvement of the M2receptor in this role depends upon the amount of cAMP elicited by the agent and the extent to which the M2 receptor opposes the increase in cAMP.

Appendix

A method for estimating the extent to which oxo-M reduced the effectiveness of the various relaxant agents is described below. In the absence of oxo-M, the relationship between the relaxant response against histamine-induced contractions (yh) and the concentration of the relaxant agent (X) can be described by the following equation of Furchgott:yh=feXRTX+KdEquation A1In equation A1, e denotes the intrinsic efficacy of the relaxant agent, RT denotes the total receptor concentration, Kd denotes the dissociation constant of the relaxant agent for its receptor andf denotes the function describing the relationship between the stimulus and the response (i.e., stimulus = occupancy × intrinsic efficacy). In the case of forskolin,Kd denotes the dissociation constant of forskolin for its site on adenylyl cyclase. In the presence of oxo-M, we propose that the relaxant response is reduced because oxo-M inhibits the cAMP accumulation elicited by the relaxant agent. Although oxo-M does not directly affect the intrinsic efficacy of the relaxant agent, it does make the agent behave as if it has reduced intrinsic efficacy. Thus, in the presence of oxo-M, the relaxant response (yo) can be described byyo=fqeX′RTX′+KdEquation A2in which X′ denotes the concentration of the relaxant agent when oxo-M is used as the contractile agent and qdenotes the apparent factor by which the intrinsic efficacy has been reduced. In the text, we refer to q as “stimulus inactivation.” To calculate q, equivalent degrees of relaxation are compared in the presence of histamine and oxo-M:feXRTX+Kd=fqeX′RTX′+KdEquation A3Rearranging and taking the logarithm of both sides yields:logX=logX′qKdKd+(1−q)X′Equation A4Nonlinear regression analysis was used to fit the equation shown above to the relaxation data to obtain estimates of q andKd. Estimates of the concentration of relaxant agent (X) were interpolated from the histamine relaxation curve that yielded responses equivalent to those generated by the relaxant agent at concentration X′ when oxo-M was used as the contractile agent. This analysis is analogous to the method of Furchgott (1966), as described previously (Ehlert, 1986).